Purpose.:
The Israeli and Palestinian populations are known to have a relatively high level of consanguineous marriages, leading to a relatively high frequency of autosomal recessive (AR) diseases. Our purpose was to use the homozygosity mapping approach, aiming to prioritize the set of genes and identify the molecular genetic causes underlying AR retinal degenerations in the Israeli and Palestinian populations.

Results.:
We recruited for the study families with AR nonsyndromic retinal degenerations, including mainly retinitis pigmentosa (RP), cone-rod degeneration (CRD), and Leber congenital amaurosis (LCA). With the aim to identify the causative genes in these families, we performed homozygosity mapping using whole genome single nucleotide polymorphism (SNP) arrays in 125 families. The analysis revealed the identification of 14 mutations, 5 of which are novel, in 16 of the families. The mutations were identified in the following eight genes: RDH12, PROM1, MFRP, TULP1, LCA5, CEP290, NR2E3, and EYS. While most patients had a retinal disease that is compatible with the causing gene, in some cases new clinical features are evident.

Conclusions.:
Homozygosity mapping is a powerful tool to identify genetic defects underlying heterogeneous AR disorders, such as RP and LCA, in consanguineous and nonconsanguineous patients. The identification of significant and large homozygous regions, which do not include any known retinal disease genes, may be a useful tool to identify novel disease-causing genes, using next generation sequencing.

Introduction

The homozygosity mapping approach was first introduced in 1987 and can be used to trace the inheritance of a chromosomal region from an ancestor through consanguineous heterozygous parents to a homozygous patient.1 Homozygous regions can be detected using microarrays that include a large number of single nucleotide polymorphisms (SNPs), which are distributed over the genome and can be genotyped simultaneously. Genomic regions that contain a significant number of consecutive homozygous SNPs are considered homozygous regions that can be either homozygous by chance or autozygous due to a shared origin; that is, an identical by descent (IBD) locus.2 Patients who are the result of consanguineous marriages and suffer from an autosomal recessive (AR) disorder are likely to be homozygous for the disease-causing mutation. The causative gene and SNPs surrounding the gene locus also are homozygous. The size of the IBD region depends on the number of generations between the ancestor who carried the recessive mutation and the homozygous patient, becoming smaller in each generation. Homozygous mutations usually are detected in patients of consanguineous parents, but also can be found in patients from relatively isolated populations, where the chance of the parents being distant relatives is high, but cannot be traced by the available family history.3–5 On the other hand, one also should bear in mind that in some cases patients of consanguineous marriages are not homozygous for the disease-causing mutation, but rather are compound heterozygous and homozygosity mapping might be misleading in such families.6–8

The Palestinian and Israeli populations include a large number of subpopulations (e.g., Jews, Arab-Muslims, Arab-Christians, Bedouin, and Druze) with a relatively high frequency of consanguineous marriages and marriages between members of relatively isolated subpopulations.9–11 The rates of consanguineous marriages are higher among Arab-Muslims, Druze, and Bedouins compared to Christian-Arabs10 and Jews.11 In more than 25% of Arab-Muslims and Druze marriages, the spouses are first cousins, with an additional 20% of the spouses being related in other ways.10 Consanguinity is less frequent among Arab-Christians (21% of marriages with first cousins and 11% related), but more frequent among the Bedouin population (35% of marriages with first cousins and 34% related).9 The Jewish population in Israel consists of a large proportion of immigrants who came to the country during the last half of the 20th century. Until the immigration of these families, many were isolated from other Jewish communities for centuries, and hence were forced to marry their distant or close relatives.11 The consanguinity rate among couples (60 years ago) varies among different ethnic groups (1.4% among Ashkenazim, 4.7% among Sephardim, and 13.6% among Eastern Jews). This pattern was maintained after the immigration, but lately, changes have been reported in the marriage patterns among Jews. The current consanguinity rate stands on 0.4% among Ashkenazim, 0.9% among Sephardim, and 2.6% among Eastern Jews.11 In addition, many Jews prefer intracommunity marriages (25% Ashkenazim, 22% Sephardim, and 17% of Eastern Jews).11

These relatively high rates of consanguinity can dramatically affect the prevalence of AR diseases. In the North American population, less than 1% of marriages are consanguineous and the percentage of patients who are homozygous for a disease-causing mutation is relatively low (schematically shown in Fig. 1A). In the Jewish and Palestinian populations, however, consanguinity levels are much higher (Figs. 1B–C) and, therefore, a relatively high number of individuals with homozygous mutations are found. This also is true for AR retinal disorders, including Leber congenital amaurosis (LCA), retinitis pigmentosa (RP), cone-rod dystrophy (CRD), and Stargardt sisease (STGD).12–16 The condition of RP is the most common inherited retinal dystrophy characterized by clinical and genetic heterogeneity. Patients with RP suffer from night blindness followed by a gradual loss of peripheral vision, usually leading to blindness.17–19 The main clinical characteristics of RP are bone spicule–like pigmentations (BSPs), atrophy of the peripheral RPE, attenuation of retinal vessels, a waxy pallor of the optic disc, and absent or severely reduced a-waves on electroretinography (ERG) tests.17

Consanguineous and intercommunity marriages in different populations in Israel compared to the North American population and their influence on inheritance of autosomal recessive diseases. (A) The North American population is large and heterogeneous, with low rate of consanguineous marriages, reducing the chances that a person will inherit two alleles that originate from the same common ancestor. (B) The different Jewish subpopulations. The two upper panels represent the time before the establishment of Israel (1948) in which most Jews lived in closed distant communities. Since the establishment of Israel (lower panel) many Jews immigrated to this country, resulting in reduced levels of consanguinity and intracommunity marriages. (C) The Arab-Muslim population (as an example for a non-Jewish population in Israel) is characterized by a very high and stable rate of consanguineous and intracommunity marriages. The population usually is divided to small villages (represented by different silhouette colors). In each panel, the population is represented by silhouettes representing the general population, while different silhouette colors represent the different subpopulations. An affected individual is represented as a dark-shaded silhouette, with the different hues represent different mutations in the population. An affected individual might be homozygous for a mutation (represented by homogeneous silhouette shade) or compound heterozygous (represented by two different hues). The percentages represent the consanguinity rates reported in each subpopulation. Arrows represent a number of generations from one panel to another.

Figure 1

Consanguineous and intercommunity marriages in different populations in Israel compared to the North American population and their influence on inheritance of autosomal recessive diseases. (A) The North American population is large and heterogeneous, with low rate of consanguineous marriages, reducing the chances that a person will inherit two alleles that originate from the same common ancestor. (B) The different Jewish subpopulations. The two upper panels represent the time before the establishment of Israel (1948) in which most Jews lived in closed distant communities. Since the establishment of Israel (lower panel) many Jews immigrated to this country, resulting in reduced levels of consanguinity and intracommunity marriages. (C) The Arab-Muslim population (as an example for a non-Jewish population in Israel) is characterized by a very high and stable rate of consanguineous and intracommunity marriages. The population usually is divided to small villages (represented by different silhouette colors). In each panel, the population is represented by silhouettes representing the general population, while different silhouette colors represent the different subpopulations. An affected individual is represented as a dark-shaded silhouette, with the different hues represent different mutations in the population. An affected individual might be homozygous for a mutation (represented by homogeneous silhouette shade) or compound heterozygous (represented by two different hues). The percentages represent the consanguinity rates reported in each subpopulation. Arrows represent a number of generations from one panel to another.

Nonsyndromic RP can be caused by mutations in one of at least 40 known genes or 7 mapped, but not identified loci (available in the public domain at https://sph.uth.edu/retnet/), some of which are known to cause other types of retinal degenerations, such as LCA and CRD. Those diseases also are heterogeneous and are known to have variable expression.4,20,21 Because of the variable expression of retinal diseases, it occasionally is difficult to distinguish between them, especially when the diagnosis is given at an advanced disease stage.

Due to the high heterogeneity level of inherited retinal diseases, complete sequence analysis of all known genes is expensive and time-consuming. An alternative method is homozygosity mapping, which was shown to be effective for gene identification mainly in consanguineous families,4,22,23 and proved to be efficient in the Israeli and Palestinian populations.12,14,16 The main goal of this study was to use homozygous mapping in Israeli and Palestinian families with retinal degenerative diseases, aiming to identify the cause of the disease. Using this method, we report the identification of 14 mutations, 5 of which are novel, in 16 families.

Methods

Subjects and Clinical Evaluation

All participants in the study signed an informed consent that adhered to the tenets of the Declaration of Helsinki before a blood sample was drawn for molecular analysis.

Genomic DNA was extracted from peripheral blood of the participants using FlexiGene DNA kit (Qiagen, Venlo, The Netherlands). DNA samples of affected patients (at least one from each family) were genotyped using different SNP microarray platforms, including Affymetrix (Santa Clara, CA) 10 K, 50 K, 250 K, and 6.0 arrays as well as Illumina (San Diego, CA) 6 K. The array data were analyzed manually using an Excel sheet (Microsoft Corporation, Redmond, WA) and the Homozygositymapper online program (available in the public domain at http://www.homozygositymapper.org/), and regions of homozygosity were identified. All homozygous regions in each family were searched for genes that were reported previously to cause a retinal disease. A homozygous region was defined as harboring at least 39 consecutive homozygous SNPs (10k arrays).2 Similarly, for 50 K, 250 K, and 6.0 arrays, a region was defined homozygous if at least 195, 975, or 3900 consecutive SNPs were homozygous, respectively.

Mutation Analysis

Sanger sequencing was performed on selected known retinal disease genes residing within homozygous regions, including ABCA4, CEP290, CERKL, CRB1, CREB3, DHDDS, EYS, FAM161A, GUCY2D, IMPG2, LCA5, MFRP, NR2E3, NRL, RDH12, TULP1, and USH2A. All exons and exon-intron boundaries of these genes were amplified by PCR. Available family members were analyzed using direct sequencing. Primers were designed using the PRIMER3 program (available in the public domain at http://frodo.wi.mit.edu/, Supplementary Table S1). A PCR was performed in a 30 μL reaction with 35 cycles. The possible pathogenicity of missense changes was evaluated using PolyPhene2 (available in the public domain at http://genetics.bwh.harvard.edu/pph2/), MutationTaster (available in the public domain at http://www.mutationtaster.org/), and SIFT (available in the public domain at http://sift.jcvi.org/). The estimated frequency of the novel mutations in the general population was examined using the following public databases: EVS (available in the public domain at http://evs.gs.washington.edu/EVS/), NCBI dbSNP (available in the public domain at http://www.ncbi.nlm.nih.gov/snp), and 1000 genomes (available in the public domain at http://browser.1000genomes.org/index.html).

Results

To determine the genetic cause underlying different retinal degenerations (including RP, CRD, and LCA), we performed whole genome homozygosity mapping in 125 families in whom at least one individual was affected. In 67 of the 125 families, homozygosity mapping revealed 1 to 5 candidate genes that were negative upon screening by Sanger sequencing. In 42 families, the number of homozygous regions was high and a large number of candidate genes were identified, but not all were screened. In 16 of the families (14 of whom are consanguineous), mutation analysis of known retinal genes that are located within homozygous regions revealed the identification of disease-causing mutations in 24 patients (Table 1). The average accumulative size of homozygous regions in patients of consanguineous marriages is 219.2 Mb, representing on average approximately 7% of the genome which is homozygous. Significant large homozygous regions were identified in all analyzed individuals, ranging from 6.7 to 120 Mb. In each family, at least one known retinal disease gene was found within significant homozygous intervals, with an average of 10.4 known genes per patient. Sequence analysis of known genes revealed the identification of 14 pathogenic mutations in eight genes (Table 1), five of which are novel. In 23 of 24 patients, the causative gene was found in one of the five largest homozygous regions (average rank of 2 and average size of 26.7 Mb).

Five mutations were novel: three null and two missense changes affecting highly conserved amino acids (Fig. 2) in TULP1 (p.D94Y) and RDH12 (p.R239Q). These missense mutations are predicted to be pathogenic by online mutation prediction softwares (Polyphene2, MutationTaster, and SIFT, see Fig. 2) and were not found in the public databases, indicating that these sequence changes are extremely rare in the general population. In addition, arginine at position 239 in the RDH12 protein was reported previously to be affected by another pathogenic missense change (p.R239W).25 The null mutations were identified in TULP1 (p.W450*), LCA5 (p.Y354*), and EYS (p.V3096Kfs*28). In most families we performed a segregation analysis and verified that the disease-causing mutation cosegregates with the phenotype (Fig. 3). The identified mutations and the resulting phenotypes are detailed below.

Evolutionary conservation and evaluation of the novel missense mutations by different online prediction programs. The arrows indicate the position of the mutated amino acid, with 10 additional surrounding amino acids (aa) on each side. The color of each aa indicates the residues physicochemical properties. The aa type is color-coded: small aa in red, acidic in blue, basic in magenta, and hydroxyl + amine + basic in green. The predicted effect of the mutation on the protein as estimated by online prediction programs, such as PolyPhen-2, MutationTaster, and SIFT, is depicted in the bottom panel. For each mutation, a very high score of pathogenicity for the altered amino acid was obtained. Accession numbers of protein sequences are as follows: TULP1- human (NP_003313.3), chimpanzee (XP_518426.2), dog (XP_538879.3), mouse (NP_067453.1), cattle (NP_001193657.1), chicken (NP_989946.1), and zebrafish (XP_005172409.1), RDH12- human (NP_689656.2), chimpanzee (XP_003314454.1), dog (XP_547866.3), mouse (NP_084293.1), cattle (NP_899207.1), chicken (XP_421193.1), and zebrafish (NP_001002325.1).

Figure 2

Evolutionary conservation and evaluation of the novel missense mutations by different online prediction programs. The arrows indicate the position of the mutated amino acid, with 10 additional surrounding amino acids (aa) on each side. The color of each aa indicates the residues physicochemical properties. The aa type is color-coded: small aa in red, acidic in blue, basic in magenta, and hydroxyl + amine + basic in green. The predicted effect of the mutation on the protein as estimated by online prediction programs, such as PolyPhen-2, MutationTaster, and SIFT, is depicted in the bottom panel. For each mutation, a very high score of pathogenicity for the altered amino acid was obtained. Accession numbers of protein sequences are as follows: TULP1- human (NP_003313.3), chimpanzee (XP_518426.2), dog (XP_538879.3), mouse (NP_067453.1), cattle (NP_001193657.1), chicken (NP_989946.1), and zebrafish (XP_005172409.1), RDH12- human (NP_689656.2), chimpanzee (XP_003314454.1), dog (XP_547866.3), mouse (NP_084293.1), cattle (NP_899207.1), chicken (XP_421193.1), and zebrafish (NP_001002325.1).

Pedigree structures of families with identified mutations. Affected individuals are indicated with filled symbols and unaffected with open symbols. Arrows indicate the index cases in each family. Double line represents consanguinity. The roman numbers on the left represent the generation number.

Figure 3

Pedigree structures of families with identified mutations. Affected individuals are indicated with filled symbols and unaffected with open symbols. Arrows indicate the index cases in each family. Double line represents consanguinity. The roman numbers on the left represent the generation number.

The RDH12 gene encodes retinol hydrogenase 12, a cytoplasmic enzyme that has an important role in the visual cycle by converting all-trans-retinal to all-trans-retinol in photoreceptors as a first step of vision.26 The RDH12 mutations are associated with LCA, early-onset RP (EORD), and CRD. Patients with RDH12 mutations show severe loss of visual acuity (VA) at an early age and severe reductions in full-field ERG amplitudes.27–30 Seven of the 16 families we analyzed in this study were found to have EORD due to either missense (6 families) or splicing (1 family) mutations in this gene (Table 1).

Family MOL0020.

A large Arab-Muslim family from East-Jerusalem with multiple consanguineous marriages and eight individuals diagnosed with CRD (Fig. 3) was recruited for the study. Six affected siblings were evaluated clinically (Table 2), and had low VA, hyperopia, and abnormal color vision. Full-field ERG testing revealed low responses with a cone > rod pattern. Homozygosity mapping performed on five patients revealed many (3–11) homozygous regions per patient, which contained 9 to 13 candidate genes (Table 1). By combining homozygosity mapping data of family members, a single large shared homozygous region of 14.9 Mb was identified, including RDH12. Sequence analysis of RDH12 revealed a reported missense mutation (p.T49M)25,28 that cosegregated with CRD in this large family. Screening of 14 additional Arab-Muslim families with retinal degeneration for this mutation was negative.

In cases where the age in which ERG data were recorded is different from that shown in the first column, the age is shown in parentheses following the ERG data. ND, not detected; NA, not available.

Family MOL0098.

A large Arab-Muslim family from East-Jerusalem with multiple consanguineous marriages and five affected individuals (Fig. 3) was recruited for the study. The index case showed typical signs of EORD, including nondetectable ERG responses (Table 2). Homozygosity mapping performed on two affected siblings revealed only one shared homozygous region (19.2 Mb) harboring RDH12. Sequencing analysis revealed a reported splice-site mutation (c.658+1G>A)29 that cosegregated with EORD in this large family. Screening of 37 additional families with retinal degeneration of the same origin was negative.

Families MOL0146 and MOL0652.

These two large multiple consanguineous families are of Bedouin and Arab-Muslim origin, respectively (Fig. 3, Table 1). Four patients were evaluated clinically and presented with EORD (Table 2). Homozygosity mapping performed on three patients revealed a number of significant homozygous regions, with an average size of 17.8 and 32.4 Mb, respectively, including RDH12. Sequence analysis of RDH12 revealed a missense mutation (p.L274P) reported in a Saudi Arabia family.27 Screening of seven additional families with EORD of the same origin was negative.

Families MOL0412, MOL0422, and MOL0659.

In three additional families (MOL0412, MOL0422, MOL0659), homozygosity mapping in one affected individual each was enough to identify the disease-causing mutation in RDH12.

Family MOL0412.

The index case of this small consanguineous Arab-Christian family (Fig. 3) was diagnosed with CRD in her childhood and at age of 15 showed low ERG responses (Table 2). Nine homozygous regions were identified in the index case (with the average size of 17.7 Mb) which contained six candidate genes, including RDH12. Sequence analysis revealed a known missense mutation (p.R161W)25 that was absent in 37 additional families.

Family MOL0422.

The index case of this consanguineous Arab-Muslim family was diagnosed with EORD at age of 17 years, and had low VA and nonrecordable ERG responses (Table 2, Fig. 3). Six large homozygous regions were identified in the index case (with an average size of 28.6 Mb) which contained eight candidate genes, including RDH12. Sequence analysis revealed a novel missense mutation in exon 6 (p.R239Q) in a conserved amino acid. This change also was predicted to be damaging by different online prediction programs (Fig. 2). Another mutation affecting the same amino acid (p.R239W) was reported previously.28 Screening of additional seven families of the same origin was negative.

Family MOL0659.

Two sibling of this consanguineous Moroccan Jewish family were diagnosed with EORD (Table 2 and Fig. 3). Homozygosity mapping performed on one individual revealed three large homozygous regions, including three candidate genes. A known missense mutation (p.L99I)25 was identified by RDH12 sequencing analysis. Screening additional 12 families of the same origin revealed two additional Jewish families originated from Tunisia and Morocco (MOL1042 and MOL1164), with the same homozygous mutation.

TULP1 Gene

The TULP1 gene encodes the tubby-like protein 1 that controls the vesicular trafficking of photoreceptor proteins in the nerve terminal during synaptic transmission and at the inner-segment during translocation of proteins to the outer segment.31,32 The TULP1 mutations cause LCA and juvenile RP.33,34 During this study we identified two novel TULP1 mutations.

Family MOL0367.

The nuclear family was a consanguineous Druze family with one affected child, diagnosed with LCA and coats-like changes treated by cryo in the left eye and by laser in both eyes (Figs. 3, 4; Table 2). Homozygosity mapping revealed 10 large homozygous regions (with an average size of 18.9 Mb) and sequencing of TULP1, one of the 14 candidate genes located in these areas, revealed a novel nonsense mutation (p.W450*). A screen of 17 additional families revealed a family (MOL1145) of the same origin in whom homozygosity for this mutation was the cause of disease.

Ocular phenotype of a patient with a homozygous TULP1 mutation and a clinical diagnosis of LCA and coats disease (MOL0367 V:2). (A–C) Color fundus (A, B) and fluorescein angiography (C) of the right eye at age 19 years (MOL0367-1) showing LCA with Coats disease. (D–F) Color fundus (D, E) and fluorescein angiography (F) of the left eye of the same patient.

Figure 4

Ocular phenotype of a patient with a homozygous TULP1 mutation and a clinical diagnosis of LCA and coats disease (MOL0367 V:2). (A–C) Color fundus (A, B) and fluorescein angiography (C) of the right eye at age 19 years (MOL0367-1) showing LCA with Coats disease. (D–F) Color fundus (D, E) and fluorescein angiography (F) of the left eye of the same patient.

A consanguineous Arab-Muslim family was noted with a single patient affected with LCA (Fig. 3). At the age of 6 years, his ERG was nondetectable (Table 2). Homozygosity mapping (HM) revealed 14 large homozygous regions (an average size of 21 Mb), with 15 candidate genes, including TULP1, that was located within the largest homozygous region. The TULP1 sequencing analysis revealed a novel missense mutation (p.D94Y) in a highly conserved amino acid. This change also was predicted to be damaging by different online prediction programs (Fig. 2). A screen of six additional families of the same origin was negative.

PROM1 Gene

The PROM1 gene encodes a pentaspan transmembrane glycoprotein that is expressed in rod and cone photoreceptors.35 The PROM1 mutations result in RP and CRD.35–37 Family MOL0256 is an Arab-Christian family with seven siblings, three of whom were diagnosed with CRD (Fig. 3). Homozygosity mapping performed on one of the patients revealed three large homozygous regions (with an average size of 16.3 Mb), including PROM1. A subsequent mutation analysis revealed a homozygous nonsense mutation (p.L386*). Two of the affected siblings were examined clinically (Table 2), and had very low VA, nystagmus, and high myopia. The mutation was negative in another family of the same origin.

MFRP Gene

The membrane-type frizzled-related protein is expressed selectively in the RPE and the ciliary body, but its exact function is unknown.38,39 Recessive mutations of MFRP cause nanophthalmos, and have been reported to be associated with an AR ophthalmic syndrome characterized by posterior microphthalmos, high hyperopia, RP, foveoschisis, and optic disc drusen.40,41 We recruited for the study an Arab-Muslim family (MOL0292) with no reported consanguinity, but the parents originated from the same village (Fig. 3). Homozygosity mapping performed on the two affected siblings revealed relatively small homozygous regions (less than 7 Mb). The third region on chromosome 11 included the MFRP gene and sequence analysis revealed a homozygous frameshift mutation (c.498delC, p.P166Pfs*26). This mutation was published previously as p.P166fsX190,41 and is predicted to result in either premature termination of the protein or absence of protein product. The patients were diagnosed with EORD, and had reduced VA and ERG amplitudes (Table 2). In addition, the two children had high hyperopia, thick retina, especially in the macula (400 and 550 μm), with a cyst or fluids under the retina and epiretinal membrane. A screen of two additional families for this mutation was negative.

LCA5 Gene

This gene encodes lebercilin, which is involved in photoreceptor ciliary function.4,42 Mutations in LCA5 cause early-onset and severe visual disturbance with nystagmus, abnormal VA, extinguished ERGs, and fundus features of severe retinal degeneration.4,43 We identified a consanguineous Arab-Muslim family (MOL0615) with one affected child who was diagnosed with LCA (Fig. 3). Nine large homozygous regions with 13 candidate genes were identified. The largest region (42 Mb) contains two candidate genes, EYS and LCA5. Screening of these two genes revealed a homozygous frameshift mutation in LCA5 (c.1062_1068del) that was absent in two additional families with LCA of the same origin.

EYS Gene

The EYS gene is the largest gene known to be expressed in the human eye; the product of this gene contains multiple epidermal growth factor (EGF)-like and LamG domains. This protein is expressed in the photoreceptor layer of the retina and localized to outer segments of photoreceptor cells.44 Considering the evolutionary data and the function of the Drosophila homolog eyes, EYS is likely to have a role in the modeling of retinal architecture.45,46 Mutations in this gene are known to cause autosomal recessive retinitis pigmentosa (ARRP).44,47,48

A consanguineous Lithuanian Jewish family (MOL0788) with two siblings affected with ARRP was recruited for the study (Fig. 3). One of the siblings had a vitreous fibrosis and bone spicules pigments in the retina, and could barely recognize hand movement (Table 2). Homozygosity mapping revealed seven large homozygous regions (with an average size of 15.8 Mb) which contain six candidate genes, including EYS. Sequence analysis revealed a novel homozygous frameshift mutation (p.V3096Kfs*28) that was found also in 2 (MOL0333 and MOL0605) of 69 families with ARRP of the same origin.

CEP290 Gene

The CEP290 gene encodes a protein that is localized in the transition zone of photoreceptors and interacts with several ciliary transport proteins in photoreceptors.49–51 Studies on silencing of CEP290 revealed an important role of CEP290 in regulating cilia assembly and vertebrate ciliary development.51 The CEP290 mutations affect photoreceptor development or maturation and cause EORP or LCA.52–54 Family MOL0226 is a consanguineous Arab-Christian family with two children affected with LCA (Fig. 3). One of the siblings was examined clinically and had extinguished ERGs (Table 2). Homozygosity mapping on both children revealed four shared homozygous regions, including six candidate genes. Mutation analysis of two genes, CERKL and CEP290, revealed a known homozygous nonsense mutation in CEP290 (p.Q1597*) that was absent in eight index patients of the same origin.

NR2E3 Gene

The NR2E3 gene (nuclear receptor subfamily 2, group E, member 3) is a photoreceptor cell-specific nuclear receptor. It is expressed only in the retina and is known to cause several retinal diseases, including enhanced S-cone syndrome (ESCS), ARRP, ADRP, clumped pigmentary retinal degeneration (CPRD), and Goldmann-Favre syndrome (GFS).55–59

Family MOL0481.

The index case of this large consanguineous Arab-Muslim family (Fig. 3) was diagnosed with juvenile RP, and had very low cone ERG responses and nonrecordable rod ERG responses (Table 2). Homozygosity mapping showed 14 large homozygous regions (with an average size of 21.7 Mb) harboring 17 candidate genes. Sequence analysis of NR2E3, located within the third largest regions, revealed a known homozygous splice-site mutation (c.118-2A>C).

Family MOL0797.

A consanguineous ARRP family of Yemenite Jewish origin with three affected siblings was recruited for this study (Fig. 3). Homozygosity mapping performed on one affected individual revealed four large homozygous regions (with an average size of 39.76 Mb) harboring nine candidate genes. A clinical reexamination of the index case raised the possibility of the ESCS diagnosis. Consequently, a deeper look at the smaller nonsignificant homozygous regions, revealed additional candidate genes, including NR2E3. The homozygous region was relatively small (5 Mb) and was not among the 10 largest homozygous regions in this individual. Sequencing analysis of NR2E3 revealed the same splice-site mutation (c.118-2A>C).

Discussion

In this study we performed homozygosity mapping using whole genome SNP arrays to identify the genetic defect in families with different AR nonsyndromic retinal degenerations. Using this approach, we successfully identified the causative gene in 14 consanguineous families and two nonconsanguineous families from genetic isolated communities. A subsequent screen for these mutations in other individuals revealed five additional families.

On average, 6% of the genome of a child of first cousins are expected to be homozygous; however, when multiple consanguineous marriages through several generations occur, homozygosity level might rise to 11%.60 Those areas become smaller as the parents are more distantly related, but long homozygous regions also may be present in individuals with unrelated parents.61,62 In general, individuals who belong to the European population, which is considered to be one of the most outbred populations, have an average 93 Mb length of total homozygous areas, which is 3.5% of the genome.62 Other studies showed that in consanguineous families, the cumulative size of homozygous regions covers 7.2% to 11% of the genome.23,60 In our cohort of patients, the average accumulative homozygous regions is 219.2 Mb, representing approximately 7% of the genome and is in line with previous reports.

In the current study, the average size of a homozygous regions containing the disease-causing mutation was 26.7 Mb, which is larger than those reported in previous studies.4,22,60,63 Large homozygous regions were identified in all analyzed individuals, although in consanguineous families the number and size of homozygous regions were significantly larger (ranging from 6.72–120 Mb, average of 25.0 Mb), than in the nonconsanguineous families (ranging from 1.4–19.6 Mb, average of 8.5 Mb), as expected. Possible explanation for the homozygous regions in the nonconsanguineous families could be that the parents have some unknown common ancestor that lived in the same village. Due to repeated consanguineous marriages in many of the families we are studying, it is expected that each patient will have many large genomic homozygous regions, while only one of those harbors the disease-causing mutation. Therefore, it is important to assess the range of homozygous regions that do not carry such mutations, and have a better prediction of the expected size and rank of such regions. In the current study, the disease-causing gene was found in one of the five largest areas in 15 of 16 families; in one family the causative gene was found in a very small homozygous region that was not even between the 10 largest regions in this patient. A similar analyses in European patients showed a higher rank, the causative mutation in 92% of the studied patients (64 of 69) was found in one of the five largest regions.3,4,22,23 This might be explained by the characteristics of the Israeli and Palestinian communities that have a tendency to marry within the family or the community along many generations. This fact leads to multiple homozygous genomic regions that might complicate homozygosity mapping studies.

Although homozygosity mapping aids to focus on specific regions in which chances of finding the genetic cause of the disease are higher, in many cases in which there are multiple consanguineous marriages along many generations, this approach yields a large number of homozygous regions, making it more difficult to concentrate on a specific causative gene. In such cases, combination of the homozygosity mapping data from a number of affected family members filter out the nonrelevant regions. For example, in three families with multiple consanguineous marriages (MOL0020, MOL0098, and MOL0146), homozygosity mapping analysis was performed on 2 to 6 affected family members, and by combining the data, we were able to exclude most of the nonshared regions (from approximately 10 regions per patient to 1–2 much shorter regions per family, including 1–2 candidate genes).

In one family (MOL0292), from a genetically isolated community, homozygosity mapping that was performed at high resolution failed to reveal any significantly large homozygous regions. The causative gene, MFRP, was found in the third largest region spanning only 3.4 Mb. This is a good example for the advantage of homozygosity mapping, which is effective even in cases when the parents are distantly related or originated from a genetically isolated community.

Another interesting case is family MOL0797 with many consanguineous marriages throughout generations, including the parents of the index case. Therefore, we expected to identify a homozygous mutation within a large homozygous region in the affected individuals. We, indeed, identified large homozygous regions, including nine known RP genes. Subsequently, a more detailed clinical analysis pointed to the causative gene, NR2E3, which is located at a very small region of 5 Mb, and was not among the 10 largest regions. In this case, using HM might be misleading, because of the tendency to focus the analysis on large homozygous areas in consanguineous families.

In rare cases, RP is associated with Coats disease, which is characterized by abnormal development of blood vessels behind the retina. So far, the only RP gene known to be associated with this combination was CRB1.64 The index case of family MOL0367 was diagnosed with LCA and Coats disease due to a novel nonsense mutation in TULP1. To our knowledge, this is the first report describing this association.

Homozygosity mapping was demonstrated in many studies as a powerful tool for identification of known potential genes for screening in patients with heterogeneous diseases. It also was shown to be an excellent tool for identification of novel disease causing genes.12,14,16,48,63 Homozygosity mapping, however, holds some disadvantages, including the possibility that the homozygous disease-causing mutations reside in relatively small homozygous regions, heterozygous mutations might be in a compound heterozygous state, and in some families only one individual is studied, providing insufficient information for locating the causative gene. Many families with retinal degenerations and unknown disease etiology, however, are not consanguineous and show no large homozygous regions by HM. Patient of such families might be compound heterozygotes for mutations in novel genes that can be identified mainly using whole exome sequencing (WES). Furthermore, the technology of next generation sequencing (NGS) can be combined with HM for more efficient identification of mutations, as recently reported in various studies in which novel genes were identified as the cause of autosomal recessive retinal degenerations.65–68

Consanguineous and intercommunity marriages in different populations in Israel compared to the North American population and their influence on inheritance of autosomal recessive diseases. (A) The North American population is large and heterogeneous, with low rate of consanguineous marriages, reducing the chances that a person will inherit two alleles that originate from the same common ancestor. (B) The different Jewish subpopulations. The two upper panels represent the time before the establishment of Israel (1948) in which most Jews lived in closed distant communities. Since the establishment of Israel (lower panel) many Jews immigrated to this country, resulting in reduced levels of consanguinity and intracommunity marriages. (C) The Arab-Muslim population (as an example for a non-Jewish population in Israel) is characterized by a very high and stable rate of consanguineous and intracommunity marriages. The population usually is divided to small villages (represented by different silhouette colors). In each panel, the population is represented by silhouettes representing the general population, while different silhouette colors represent the different subpopulations. An affected individual is represented as a dark-shaded silhouette, with the different hues represent different mutations in the population. An affected individual might be homozygous for a mutation (represented by homogeneous silhouette shade) or compound heterozygous (represented by two different hues). The percentages represent the consanguinity rates reported in each subpopulation. Arrows represent a number of generations from one panel to another.

Figure 1

Consanguineous and intercommunity marriages in different populations in Israel compared to the North American population and their influence on inheritance of autosomal recessive diseases. (A) The North American population is large and heterogeneous, with low rate of consanguineous marriages, reducing the chances that a person will inherit two alleles that originate from the same common ancestor. (B) The different Jewish subpopulations. The two upper panels represent the time before the establishment of Israel (1948) in which most Jews lived in closed distant communities. Since the establishment of Israel (lower panel) many Jews immigrated to this country, resulting in reduced levels of consanguinity and intracommunity marriages. (C) The Arab-Muslim population (as an example for a non-Jewish population in Israel) is characterized by a very high and stable rate of consanguineous and intracommunity marriages. The population usually is divided to small villages (represented by different silhouette colors). In each panel, the population is represented by silhouettes representing the general population, while different silhouette colors represent the different subpopulations. An affected individual is represented as a dark-shaded silhouette, with the different hues represent different mutations in the population. An affected individual might be homozygous for a mutation (represented by homogeneous silhouette shade) or compound heterozygous (represented by two different hues). The percentages represent the consanguinity rates reported in each subpopulation. Arrows represent a number of generations from one panel to another.

Evolutionary conservation and evaluation of the novel missense mutations by different online prediction programs. The arrows indicate the position of the mutated amino acid, with 10 additional surrounding amino acids (aa) on each side. The color of each aa indicates the residues physicochemical properties. The aa type is color-coded: small aa in red, acidic in blue, basic in magenta, and hydroxyl + amine + basic in green. The predicted effect of the mutation on the protein as estimated by online prediction programs, such as PolyPhen-2, MutationTaster, and SIFT, is depicted in the bottom panel. For each mutation, a very high score of pathogenicity for the altered amino acid was obtained. Accession numbers of protein sequences are as follows: TULP1- human (NP_003313.3), chimpanzee (XP_518426.2), dog (XP_538879.3), mouse (NP_067453.1), cattle (NP_001193657.1), chicken (NP_989946.1), and zebrafish (XP_005172409.1), RDH12- human (NP_689656.2), chimpanzee (XP_003314454.1), dog (XP_547866.3), mouse (NP_084293.1), cattle (NP_899207.1), chicken (XP_421193.1), and zebrafish (NP_001002325.1).

Figure 2

Evolutionary conservation and evaluation of the novel missense mutations by different online prediction programs. The arrows indicate the position of the mutated amino acid, with 10 additional surrounding amino acids (aa) on each side. The color of each aa indicates the residues physicochemical properties. The aa type is color-coded: small aa in red, acidic in blue, basic in magenta, and hydroxyl + amine + basic in green. The predicted effect of the mutation on the protein as estimated by online prediction programs, such as PolyPhen-2, MutationTaster, and SIFT, is depicted in the bottom panel. For each mutation, a very high score of pathogenicity for the altered amino acid was obtained. Accession numbers of protein sequences are as follows: TULP1- human (NP_003313.3), chimpanzee (XP_518426.2), dog (XP_538879.3), mouse (NP_067453.1), cattle (NP_001193657.1), chicken (NP_989946.1), and zebrafish (XP_005172409.1), RDH12- human (NP_689656.2), chimpanzee (XP_003314454.1), dog (XP_547866.3), mouse (NP_084293.1), cattle (NP_899207.1), chicken (XP_421193.1), and zebrafish (NP_001002325.1).

Pedigree structures of families with identified mutations. Affected individuals are indicated with filled symbols and unaffected with open symbols. Arrows indicate the index cases in each family. Double line represents consanguinity. The roman numbers on the left represent the generation number.

Figure 3

Pedigree structures of families with identified mutations. Affected individuals are indicated with filled symbols and unaffected with open symbols. Arrows indicate the index cases in each family. Double line represents consanguinity. The roman numbers on the left represent the generation number.

Ocular phenotype of a patient with a homozygous TULP1 mutation and a clinical diagnosis of LCA and coats disease (MOL0367 V:2). (A–C) Color fundus (A, B) and fluorescein angiography (C) of the right eye at age 19 years (MOL0367-1) showing LCA with Coats disease. (D–F) Color fundus (D, E) and fluorescein angiography (F) of the left eye of the same patient.

Figure 4

Ocular phenotype of a patient with a homozygous TULP1 mutation and a clinical diagnosis of LCA and coats disease (MOL0367 V:2). (A–C) Color fundus (A, B) and fluorescein angiography (C) of the right eye at age 19 years (MOL0367-1) showing LCA with Coats disease. (D–F) Color fundus (D, E) and fluorescein angiography (F) of the left eye of the same patient.